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Dominant-negative and targeted null in the endothehal receptor tyrosine kinase, tek, reveal a critical role in vasculogenesis of the embryo

Daniel J. Dumont, 1's'6 G4rard Gradwohl, 2 Guo-Hua Fong, 1 Mira C. Puri, 1'3 Marina Gertsenstein, 1 Anna Auerbach, 1 and Martin L. Breitman 1'3'4

~Division of Molecular and Developmental Biology, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada; 2Laboratoire de G6n&ique Mol&ulaire des Eucaryotes, Centre National de la Recherche Scientifique (CNRS), Unit6 184 de Biologie Mol6culaire et de G4n6tique, Institut National de la Sant6 et de la Recherche M4dicale (INSERM), Institut de Chimie Biologique, Facult6 de M6decine, Strasbourg, France; 3Department of Molecular and Medical Genetics, University Toronto,Toronto, Ontario, Canada

The receptor tyrosine kinases (RTKs) expressed on the surface of endothelial cells are likely to play key roles in initiating the program of endothelial cell growth during development and subsequent vascularization during wound healing and tumorigenesis. Expression of the Tek RTK during mouse development is restricted primarily to endothelial cells and their progenitors, the angioblasts, suggesting that Tek is a key participant in vasculogenesis. To investigate the role that Tek plays within the endothelial cell lineage, we have disrupted the Tek signaling pathway using two different genetic approaches. First, we constructed transgenic mice expressing a dominant-negative form of the Tek receptor. Second, we created a null of the tek by homologous recombination in embryonic stem (ES) cells. Transgenic mice expressing dominant-negative of Tek or homozygous for a null allele of the tek both died in utero with similar defects in the integrity of their endothelium. By crossing transgenic mice that express the lacZ reporter gene under the transcriptional control of the endothelial cell-specific tek promoter, we found that the extraembryonic and embryonic vasculature was patterned correctly. However, homozygous tek embryos had -30% and 75% fewer endothelial cells at day 8.5 and 9.0, respectively. Homozygous null embryos also displayed abnormalities in heart development, consistent with the conclusion that Tek is necessary for endocardial/myocardial interactions during development. On the basis of the analysis of mice carrying either dominant-negative or null mutations of the tek gene, these observations demonstrate that the Tek signaling pathway plays a critical role in the differentiation, proliferation, and survival of endothelial cells in the mouse embryo. [Key Words: Receptor tyrosine kinase; endothelial cell growth; Tek; vasculogenesis; transgenic mice; homozygous embryos] Received June 1, 1994; revised version accepted July 7, 1994.

The receptor tyrosine kinase {RTK) family of cell-sudace mouse all depend on intact signaling pathways that are is known to play key roles in cell-cell commu- controlled by different members of the RTK family nication in multicellular metazoan organisms (Pawson (Reith and Bernstein 1991; Pawson 1993}. and Bemstein 1991). Genetic and biochemical studies on Five RTKs have been identified whose expression is this large family of proteins have shown that different restricted primarily to cells of the endothelial lineage. RTKs are responsible for transducing important develop- On the basis of similarities in their primary amino acid mental, proliferative, cell survival, and migratory signals sequences, they have been placed into two distinct sub- from the outside to the inside of the cell {Hunter and families of RTKs (Dumont et al. 1993; Hunter and Lind- Lindberg 1994). The development and proper functioning berg 1994). The members of one subfamily, including of cell systems as diverse as the compound eye in the fly, Flt-4 (Pajusola et al. 1992) and the Vascular endothelial the vulva in the nematode, and hematopoiesis in the growth factor (VEGF) receptors Flt-1 (Matsushime et al. 1987; Shibuya et al. 1990) and Flk-1/KDR {Terman et al. 1991; Millauer et al. 1993), are characterized by extra- 4This manuscript is dedicated to the memory of Dr. Martin L. Breitman, cellular domains consisting of seven immunoglobulin- who died on February 13, 1994. SCorresponding author. like loops and an intracellular split kinase domain. The 6present address: AMGEN Institute, Toronto Ontario MSG 2C1, Canada. second subfamily contains the two receptors Tek and

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Tie (Wilks 1989; Dumont et al. 1992, 1993; Partanen et pathway by expressing a dominant-negative form of Tek al. 1992; Iwama et al. 1993; Maisonpierre et al. 1993; in transgenic mice. Previous studies have shown that Sato et al. 1993; Ziegler et al. 1993), both of whom have structural mutations that abolish or diminish the intrin- complex extracellular domains consisting of two immu- sic tyrosine kinase activity of other RTKs act in a dom- noglobulin-like loops separated by three EGF-like re- inant-negative manner in both cells and in the intact peats that are followed by three fibronectin type III-like organism. For example, naturally occurring mutations in repeats. The intracellular portions of Tek and Tie also the Kit RTK in the dominant white-spotting (W) mouse contain a split kinase domain. mutant act in a dominant-negative manner to inhibit The differing spatial and temporal patterns of expres- melanogenesis, hematopoiesis, and gametogenesis sion of these RTKs during development suggest that they (Reith et al. 1990). Similarly, expression of other RTKs play different roles within the endothelial cell lineage. with mutations that reduce their intrinsic tyrosine ac- Very early in development [embryonic day 7.0 (E7.0)], tivity act in a dominant-negative fashion to inhibit the ilk-1 is expressed in both the extraembryonic and em- signaling through the corresponding wild-type receptor bryonic mesoderm in regions thought to give rise even- in mammalian cell lines (Reith et al. 1993), in frog em- tually to the embryonic vasculature (Millauer et al. bryos (Amaya et al. 1991), in transgenic mice (Ray et al. 1993; Yamaguchi et al. 1993; D. Dumont, G.-H. Fong, 1991), and in tumor angiogenesis (Millauer et al. 1994). M.C. Puri, G. Gradwohl, K. Alitalo, and M. Breitman, in Second, we created a tek null allele by homologous re- prep.), tek can then be detected in regions that overlap combination in ES cells and introduced the with the flk-l-positive extraembryonic mesoderm at into the mouse germ line. Mice heterozygous for the E7.5, and finally, tie is detected at E8.0 in the flkol- and mutant allele were phenotypically normal, whereas ho- tek-expressing extraembryonic mesoderm (D. Dumont, mozygous mice died in utero at -E9.5 as a consequence G.-H. Fong, M.C. Puri, G. Gradwohl, K. Alitalo, and M. of an underdeveloped vasculature. Embryos for the null Breitman, in prep.). The early onset of ilk-1 expression mutation or hemizygous for the dominant-negative (E7.0) suggests a role for this receptor early in the deter- transgene displayed strikingly similar developmental de- mination of the endothelial cell lineage (Yamaguchi et fects. Taken together, these results demonstrate that the al. 1993), whereas the later, sequential onset of both tek Tek signaling pathway plays a critical role in the vascu- and tie expression suggests that these RTKs may play larization of the mouse embryo. distinct roles during subsequent vascularization of the embryo. Little is known about the expression of fit-1 and fit-4 Results in the mouse. One report has stated that fit-1 is ex- Generation of transgenic mice carrying a tek cDNA pressed in the endothelium of E9.5 embryos (Peters et al. encoding a dominant-negative Tek RTK 1993), but as the onset of expression was not determined in these studies, it is not known whether fit-1 may also To assess rapidly the role of the Tek signaling pathway play an earlier role during vascularization. Although the in mouse development, we introduced a mutation localization and timing of fit-4 expression in the mouse within the tek cDNA that altered the codon for lysine- has not been reported, studies on human fetal tissue 853 to encode an alanine residue. This lysine residue and have reported FLT-4 expression in the epithelium lining its surrounding amino acids are found in a region within the bronchioles of the lung and in the vascular endothe~ the intracellular cytoplasmic domain that is highly con- lium (Pajusola et al. 1992; Kaipainen et al. 1994). Quek- served in all tyrosine kinases, and alteration of this res- 1, the quail homolog of ilk-l, is expressed, as in the idue is known to abolish catalytic function (Nocka et al. mouse, in the mesoderm from the onset of gastrulation, 1990; Reith et al. 1990, 1993). Furthermore, other RTKs whereas the expression of Quek-2, a distinct RTK related that are competent to bind ligand, but lack intrinsic ki- most closely to fit-4, is found later in endothelial cells nase activity, act in a dominant-negative fashion to in- that stain with the endothelial cell-specific antibody hibit signal transduction by wild-type receptor coex- QH1 (Eichmann et al. 1993). pressed in the same cell (Amaya et al. 1991; Ray et al. Our studies have focused on the biological function of 1991; Reith et al. 1993; Millauer et al. 1994). To deter- the routine RTK, Tek, first identified from a cDNA li- mine whether the lysine-to-alanine mutation at codon brary prepared from E12.5 embryonic heart (Dumont et 853 affected the intrinsic tyrosine kinase activity of Tek, al. 1992, 1993). The tek gene encodes a 140-kD we introduced this mutated tek cDNA (tek A8s3) into with intrinsic tyrosine kinase activity (Dumont et al. COS cells and analyzed extracts from these cells for Tek 1993; Ziegler et al. 1993) that is expressed in the endo- activity. As shown in Figure 1, Tek A8s3 protein was cat- thelium of the embryo, in embryonic stem (ES) cells in- alytically inactive in autophosphorylation and phospho- duced to differentiate in vitro (Yamaguchi et al. 1993), in rylation of exogenously added substrate (Fig. 1B; data not endothelial cell lines (Dumont et al. 1993), and in early shown). Moreover, the engineered mutation did not alter Sca +, c-kit +, lin- hematopoietic stem cell populations the length of the protein as judged by its gel mobility (Iwama et al. 1993). To understand the role of the Tek (Fig. 1B). RTK in mouse development, we have taken two genetic Previous studies by our group and others have sug- approaches to disrupt the Tek signaling pathway in vivo. gested that the f~-actin, polyoma, and tek promoters were The first strategy was to interfere with the Tek signaling good candidates to drive expression of the tek A8s3 cDNA

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Vascular defects in tek-deficient mice

Figure 1. Tek dominant-negative trans- genes. (A} Schematic showing the trans- genes used to drive the expression of the dominant-negative mutant tek Ass3 cDNA. The solid rectangle represents the pro- moter region for each transgene; the splice donor (SD1 and acceptor (SA} of the [3-actin promoter are indicated; the immunoglob- ulin (Igl-, epidermal growth factor {EGF)-, and fibronectin type III-like {FN) repeats found in the extracellular region of Tek are depicted by hatched, stippled, and open boxes, respectively; the smaller solid box represents the transmembrane region (TM); the two kinase domains (TK1 and TK2) are depicted by open boxes separated by the kinase insert (KII; ovals at the end of each transgene represent the different viral polyadenylation sequences. The oval above TK1 represents the position of the Lys--> Ala-853 mutation. (B) TekAssa is catalytically inactive. Both the tek ASsa and wild-type tek cDNAs were expressed in COS cells using the mammalian expres- sion vector pECE. Transfected COS cells were labeled metabolically with [aSS]me- thionine and immunoprecipitated with anti-Tek antiserum. The immunoprecipi- tates were split and a portion used in an in vitro kinase assay (two left lanes); whereas the other was electrophoresed in a gel sim- ilar to the one used to analyze the products of the kinase assay, but after electrophore- sis the gel was processed for fluorography (two right lanes). (DN) Dominant-negative mutant tekASS3; (WT) wild-type tek cDNA.

within the endothelial cell lineage of transgenic mice embryos isolated on E9.5 had an enlarged pericardial cav- {Fig. 1A} (Bautch et al. 1987; Williams et al. 1988; Dubois ity and contained few blood cells in the vessels of the et al. 1991; M. Puff, D. Dumont, and M. Breitman, in yolk sac. This could be caused by hemorrhaging into the prep). yolk sac cavity, as primitive red blood cells were ob- served there. Furthermore, 5 of 19 transgenic polyoma promoter-tek Assa embryos exhibited a developmental tek ASs3 transgenic mice are delayed developmentally delay (Table 1}. Of these delayed embryos, and exhibit a defect in their endothelium two appeared to have arrested early in development On the basis of the assumption that tel< may play a crit- around day 8.0 as judged by the closure of their neural ical role in the endothelial cell lineage, we removed folds. The three other embryos were delayed in their de- transgenic founder embryos on days 9.5 and 10.5 of ges- velopment to varying levels but appeared morphologi- tation, 2-3 days after the onset of tek expression {Du- cally normal when compared with embryos of the same mont et al. 1992, and in prep.). As shown in Table 1, size. One embryo was developmentally arrested but embryos transgenic for the f3-actin-tek ^ssa transgene proved to be negative for the presence of the transgene by showed no discernible phenotype. In contrast, two of six PCR. This embryo was an amorphous mass that was transgenic embryos containing the tek promoter-tek Ass3 undergoing resorption, suggesting that its development transgene were delayed or arrested in their development was arrested before the onset of tek expression and, thus, {Table 1; data not shown}. Interestingly, one of these was considered to be phenotypically distinct.

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Table 1. Delayed development among Tek ASs3 dominant-negative transgenic embryos Total embryos Total transgenic Total development Transgene recovered (TG) delayed (DD) DD-TG/DD Polyoma tek A853 126 19 6 a 5b/6 tek-tek Ass3 64 6 2 a 2/2 f3-Actin-tek Assa 20 6 0 -- "All embryos were obtained for analysis of E9.5, except two were discovered with the polyoma-driven transgene on E10.5 and one with the tek promoter on E10.5. bOne embryo comprised a small amorphous mass of necrotic cells that was undergoing resorption at the time of assay. As such, it was considered to be phenotypically distinct from the group of transgenic embryos showing the Tek ass3 dominant-negative phenotype.

Histological analysis of the developmentally delayed the number and complexity of the branching structures transgenic embryos was carried out on all tel

Figure 2. Histological examination of the heart regions from dominant-negative tek A853 transgenic and tekaSp/+ heterozygous and homozygous embryos. E9.5 transgenic embryos, containing the tek A853 transgene were driven by either the tek promoter (A) or the polyoma early sequences {B). tekaSP/+ heterozygous (C} and homozygous (D) embryos are shown, tekaSP/+ heterozygous embryos (C} showed normal (arrowheads), whereas transgenic tek promoter (A) and polyoma promoter-tek A853 (B) and the tekaSP/+ homozygous (D) embryos showed degenerating endothelium (arrows) within their heart regions. All sections are photographed at the same mag- nification. Bar, 10 ixm.

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Vascular defects in tek-deficient mice subtle abnormalities could not be excluded. In contrast, gues that the observed phenotypes for both the tek- and the embryo used to produce the section in Figure 2B polyoma promoter-driven transgenes were intrinsic to a represented the most extreme phenotype where a defect defect in the vascular endothelium. could be distinguished clearly after dissection [data not shown). Thin sections of the heart depicted a pathology Disruption of the tek gene in ES cells and germ-line virtually indistinguishable from that observed for tek asp- transmission of the mutation targeted homozygous mutant embryos (Fig. 2D; see be- low). The development and number of trabeculae within To create a null allele of tek, we deleted the last 52 bp of this polyoma promoter-tek A8sa and tekaSP-targeted ho- exon 1 {Fig. 3A), encoding the first 17 amino acids of Tek, mozygous mutant hearts was severely reduced and my- by homologous recombination in ES cells. This deletion ocardial development seemed to be affected adversely removes both the start of translation and the signal pep- (Fig. 2B, D). The endothelial cells of the endocardium tide. Therefore, we refer to this mutant as tek asp. A pos- were few in number and not closely associated with the Rive/negative-type targeting vector (Mansour et al. myocardium. In addition, the endothelial cells had small 1993) was engineered by cloning 7.2 kb of 5' genomic granules on their surfaces, which may be calcium depos- sequence upstream of a bacterial neomycin (neo) cassette its indicating cell death or cellular degeneration. (Tybulewicz et al. 1991) and 0.7 kb of 3' genomic se- No other overt phenotype was observed for any of the quences downstream. In two separate experiments, lin- tek ASsa dominant-negative embryos, demonstrating that earized targeting vector was electroporated into R1 ES expression of this protein in other cellular compart- cells (Nagy et al. 1993). A properly targeted event was ments had no effect. Moreover, the fact that a phenotype observed (Fig. 3B; data not shown) by Southern blot with was seen with the endothelial-specific tek promoter ar- both 3' external and internal probes. Two independent

Figure 3. Disruption of the tek locus and Southern blot analysis of wild-type, tek asp heterozygous and homozygous DNA. (A) Schematic showing the strategy used to disrupt the coding sequences of the first exon of the tek gene, generating the mutation tek asp. The solid rectangle represents the protein-coding sequences; the open rectangle represents the untranslated {UT) sequences. The phosphoglyc- erate kinase {PGKj-neo expression cassette, represented by a hatched rectangle, was inserted in the same transcriptional orientation as the tek gene. The stippled rectangle represents the PGK-tk (thymidine kinase} expression cassette fused to plasmid sequences represented by small, open-ended boxes. The XbaI and EcoRI restriction sites are not indicated 5' of the first exon. The brackets around the 5' Asp718I site signify that the site was destroyed as a consequence of cloning. The location of the 3' external probe is indicated by a solid box beneath the predicted targeted locus. (B) DNA extracted from day 9.5 embryos from a tekAsp/+ hetero- zygous F1 intercross. (Trg) The tekaSP-specificfragment. The number of wild-type ( + / + ), heterozygous ( + / - ), and homozygous (- / -) embryos {2, 4 and 3, respec- tively} were at the predicted Mendelian frequency.

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ES cell lines carrying the tek asp allele were injected into (Fig. 4F) than heterozygous littermates (Fig. 4C). Vascu- host C57BL/6J blastocysts to generate chimeras that lar hemorrhaging of homozygous embryos could also be transmitted the mutation to their offspring. detected histologically when the trunk region was exam- ined. Primitive blood cells could be seen throughout the body of the embryo distributed among the mesenchymal tek asv homozygous mice die during gestation cells (Fig. 4B,E). The dorsal aorta in heterozygous em- F1 intercrosses of tekAsp/+ mice produced no live off- bryos was well defined with endothelial cells lining the spring homozygous for the tek a~p allele (Table 2); there- lumen of the vessel, and there was no blood in the trunk fore, mothers from these intercrosses were sacrificed and (Fig. 4B). In contrast, in homozygous embryos the endo- embryos were genotyped. At E9.5, some embryos from thelium of the dorsal aorta was disorganized and ap- the heterozygous cross were visibly defective, showing peared to have ruptured, resulting in blood cells in the some signs of necrosis, and their hearts were not beating. body (Fig. 4E). This localized hemorrhaging of the em- These embryos were all homozygous for the tek asp mu- bryonic vasculature most likely results in a decrease in tation (Table 2). No live homozygous mutant embryos the embryonic blood pressure that may explain the ac- were found beyond E9.5 (Table 2). At E12.5, none of the cumulation of blood in the yolk sac vasculature and em- embryos {0/35) were tek a~p homozygotes; however, bryonic portion of the placenta (Fig. 4D; data not shown). there were eight severely necrosed implantations, sug- This region of the placenta also had very few endothelial gesting that tek asp homozygous embryos implanted but cells in the sinuses of the placenta as compared with then died. Genotyping of embryos isolated on E9.5 dem- a heterozygous littermate (Fig. 4A, D). These results onstrated that the proportion of embryos that were wild clearly demonstrate that tekaSP/tek asp embryos have a type, heterozygous, and homozygous for the tek asp allele striking deficiency in the endothelium, resulting in hem- followed the expected Mendelian frequency, confirming orrhaging and pooling of blood in body cavities. that Tek is not required for implantation of the embryo The hearts of tek asp homozygous embryos were se- (Table 2). verely underdeveloped (see Fig. 2D). The myocardium of E9.5 mutant embryos did not possess a detailed organi- zation of trabeculae, and the overall growth of the myo- Hemorrhaging of tekasp/tek a+p embryos cardium seems to be reduced. Furthermore, fewer endo- Day 8.5 embryos homozygous for the tek Asp mutation thelial cells were seen in the endocardium (see Fig. 2D). were readily discernible by the grossly abnormal mor- phology of their yolk sacs, which were engorged with blood and had a cobblestone-like appearance (Figs. 4F Analysis of ilk-l, tek, and tie expression in tek asp and 5). To date, all embryos with this morphologically embryos distinct yolk sac that have been genotyped have been That there were few remaining endothelial cells in ho- homozygous tek a+p (9/9). Histological analysis of the mozygous embryos was confirmed by RNA in situ hy- yolk sacs from homozygous embryos harvested on E9.5 bridization of sections prepared from both tek Asp ho- revealed that the blood vessels in the yolk sac appeared mozygous and heterozygous embryos with a ilk-1 an- distended and were very often packed with blood (Fig. tisense riboprobe. Both heterozygous and homozygous 4F; data not shown). Furthermore, several yolk sacs iso- embryos (data not shown) contained flk-l-positive cells lated from homozygous embryos contained little or no organized in a distinctive vascular network. However, blood (Fig. 4F). Before dissection of these embryos, how- the ilk-l-positive cells in homozygous mutant embryos ever, blood could be detected in the yolk sac cavity, in- were present in discontinuous chains, suggesting that dicating that the lack of blood in the yolk sac vasculature the vessels contained a sparsely populated endothelium was caused by hemorrhaging. In addition, the yolk sac (data not shown). Moreover, the levels of ilk-1 expression vessels contained considerably fewer endothelial cells were lower in the homozygous mutants. Adjacent sec- tions probed for the expression of tek and tie demon- strated that tie transcripts were present, albeit at lower Table 2. Genotypes of progeny of F~ intercrosses of teka~p/+ levels than in heterozygous embryos (data not shown), heterozygous mice whereas no tek signals could be detected in homozygous tek asp embryos (data not shown). These results demon- Genotypes strate that the tek asp mutant allele does not produce a neonates E9.5 normal transcript, confirming that it is a null allele. In- terestingly, these results also demonstrate that tie ex- Clone +/+ +/- -/- +/+ +/- -/- pression in endothelial cells is not dependent on prior 24 108 57 0 9 6 7 expression of tek. 19 11 4 0 3 1 1 Total 119 61 0 12 7 8 tekaSP/tek a~p embryos have a reduced number Genotyping was carried out by Southern analysis on DNA ex- of endothelial cells tracted from tails or from the dissected head of embryos (see Materials and methods; Fig. 3B). To follow the fate of tek-expressing endothelial cells in

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Figure 4. Histological analysis of homozygous tek mutant embryos and normal littermates. Sections through the embryonic portion of the placenta from tek asp heterozygous (A} and homozygous {D} embryos showing the accumulation of fetal blood cells in the placental sinuses in homozygous embryos. These sections also illustrate the decreased number of endothelial cells in the sinus of mutants as compared with normal littermates (arrowheads). Bar, 10 ~m. Thin sections taken through the dorsal aortic region of heterozygous (B) and homozygous (E) embryos showing the collapsed aorta (da) and extravasated blood (arrows). Bar, 30 ~m. Stained thin sections through the yolk sac of tek asp heterozygous (C) and homozygous (F) embryos showing the distended yolk sac vessels and the decreased number of endothelial cells lining the yolk sac vessels (arrowheads). Bars, 30 ~m. mutant embryos, we crossed a tek promoter-lacZ trans- compared with their normal littermates (Fig. 7A}. This gene gene onto the tek asp mutant background. Adult decrease in cell number and staining intensity was even mice bearing the tek promoter-lacZ, tekaSP/+ genotype more accentuated in sections taken from E9.0 homozy- were then used to generate homozygous embryos carry- gous embryos (Fig. 7B,D). The blood islands also con- ing the tek asp mutation and the transgene. The tek pro- tained cells with an endothelial cell-like morphology moter-lacZ transgenic line used in these studies ex- that did not stain blue, whereas this was never observed presses the lacZ reporter gene in a manner that virtually in normal transgenic mice. recapitulates the endogenous tek expression profile Table 3 summarizes the number of blue endothelial (M.C. Puri, D. Dumont, and M. Breitman, in prep.). cells found in the yolk sacs of transgenic embryos. The On the basis of f~-galactosidase ([3-gal) activity, tek asp number of endoderm cells found in each blood island did homozygous embryos isolated on E8.5 and E9.0 con- not vary significantly for any of the embryos and, thus, tained a normally patterned vasculature in both extra- was used to normalize the values. Day 8.5 tek asp ho- embryonic and embryonic tissues (Fig. 5}. Moreover, the mozygous embryos possessed -30% fewer endothelial size of normal and homozygous embryos at these gesta- cells within the blood islands as compared with their tional ages were the same (data not shown), suggesting normal littermates. On E9.0, one-half day later in devel- that the growth of the embryo up to E9.0 is not depen- opment, 75% fewer endothelial cells were detected in dent on Tek. However, it is clear that the level of [3-gal the yolk sac of tek asp homozygous mutant embryos. staining in these homozygous embryos was reduced (Fig. These results clearly demonstrate that the number of 5C, D). Histological examination of E9.0 homozygous endothelial cells present within homozygous embryos embryos confirmed that proper patterning of the vascu- at the times analyzed is significantly lower than that lature was initiated (Fig. 6). Furthermore, the endocar- of their normal littermates and that as development dium and other vascular structures of mutant embryos progresses the number of endothelial cells decreases. formed correctly but contained only low levels of lacZ Moreover, the very low levels of lacZ expression de- expression, in keeping with the low levels of flk-1 and tie tected in many cells suggests that these cells are proba- expression detected in these cells {Fig. 6B,D; data not bly compromised metabolically and are dying. shown). Histological analysis of the yolk sacs of tek asp ho- mozygous mutants revealed that the number of lacZ- Discussion expressing endothelial cells lining the blood islands was reduced in E8.5 tek asp homozygous embryos (Fig. 7C) In this study we have demonstrated that embryos ho-

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Figure 5. The yolk sac vasculature of tek asp homozygous embryos contains fewer endothelial ceils, tek promoter-lacZ transgene expression in day 8.5 normal (A) and tek asp homozygous (C) embryos shows a reduced number of blue-staining endo- thelial ceils in the homozygous mutants. The decreased number of blue cells {arrow- heads) is even more dramatic in the yolk sac of day 9.0 tek asp homozygous {D) em- bryos, as compared with normal embryos (B). Bar, 50 lam.

mozygous for a null mutation in the tek RTK gene or not absolutely required for the elaboration of the endo- expressing dominant-negative forms of the Tek receptor thelial cell lineage but is necessary for the subsequent die in utero, with visible abnormalities in their endothe- expansion of this lineage as the embryo grows in size. lium, compromised heart development, and hemorrhag- The finding that homozygous tek asp embryos survive to ing. These data provide definitive genetic evidence that day 8.5 suggests that the number of endothelial cells the Tek RTK, whose expression is almost exclusively produced by this Tek-independent pathway can support restricted to cells of the endothelial cell lineage (Dumont growth of the embryo only up to this size, after which et al. 1992 and in prep.), is critical for proper vasculature the integrity of the vasculature is compromised, leading development. to hemorrhaging and embryonic death. The loss of endothelial cells in tek asp homozygous em- bryos after E8.5 may also be an active process if Tek is Tek is required for maintenance and/or proliferation responsible for transducing a survival stimulus. Thus, of endothelial cells, not for the production Tek-deficient endothelial cells may embark on a pro- of angioblasts and differentiation into endothelial cells grammed cell death or apoptotic pathway. The lower lev- The vasculature of the embryo has its origins in both the els of lacZ-expressing cells in the cross described here, as extraembryonic and embryonic mesoderm. Mesenchy- well as the lower number of flk-1- and tie-positive cells mal cells differentiate into angioblasts that then give rise in homozygous embryos, suggest that perhaps this may to endothelial cells (Noden 1989, 1991; Coffin et al. be the case and that these cells die selectively in the 1991). tek asp homozygous embryos have multiple signs absence of an intact Tek-signaling pathway. of vascular hemorrhaging, either because endothelial cells fail to develop in these embryos or develop and then Interaction of the endothelium and the myocardium die because of the absence of Tek. We were able to ad- during cardiogenesis dress this question by following the fate of tek-express- ing cells in embryos homozygous for the tek mutation Apart from vascular hemorrhaging, the most striking and carrying a IacZ reporter transgene driven by the en- phenotype detected in tek aSp homozygous mutant em- dothelial cell-specific tek promoter. These experiments bryos was an underdeveloped heart. The primitive myo- showed that the relative number of endothelial cells de- cardial and endocardial rudiments of the murine heart creased as development progressed, arguing that Tek is develop in situ from the pericardial wall and subadjacent

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Vascular defects in tek-deficient mice

Figure 6. The embryonic vasculature of tek asp homozygous embryos contain fewer endothelial cells. The trunk (A,C} and heart (B,D) regions of a Eg.0 tek asp homozygous (A,B) and wild-type (C,D) embryos. A lower levels of lacZ expression is seen in the intersegmental vessels (is) and endocardium (e) of mutants. (da) Dorsal aorta. Bars, 50 ~m.

mesenchyme, respectively (Kaufman and Navaratnam result either because Tek is a ligand for another receptor 1981). These two structures are thought to interact dur- present in the myocardium or because Tek can act ing differentiation of the heart, and one of the products of directly as an adhesion molecule to mediate this inter- this interaction is the cardiac jelly found between the action. If these possibilities are correct, one would pre- endocardium and the myocardium (Kaufman and Navar- dict that the catalytic function of Tek would not be re- atnam 1981). tek is expressed in the angioblasts (Du- quired for these interactions; however, the very similar mont et al. 1992) that arise from the mesenchyme and phenotype of the Tek dominant-negative (Tek-DN) mu- give rise to the endothelium of the embryo and to the tants argues that catalytic activity is required for Tek endocardium (Kaufman and Navaratnam 1981). Very function. early during development (E8.5), these structures were present in both the tek asp homozygous and wild-type Tyrosine kinase activity is required for Tek function embryos, suggesting that, although tek is expressed in these cells, the early differentiation processes leading to In these studies we have shown that expression of a Tek- these structures are Tek independent. Moreover, later in DN molecule in transgenic mice results in developmen- development (E9.5), the myocardium was severely re- tal delay and/or arrest. Histological analysis revealed tarded in growth, and the endocardium, which also had that several of the embryos had abnormalities in their an abnormal appearance, was not associated closely with endothelium, including fewer endothelial cells, which the myocardium supporting a role for endocardial/myo- were apparently degenerating. cardial interactions later in heart development. How- This phenotype was very similar to embryos with a ever, we cannot exclude the possibility that the abnor- targeted null mutation, tek asp, which had fewer endo- mal myocardial development is secondary to the endo- thelial cells, compromised heart development, and hem- thelial cell defect. orrhaging. The extracellular domain of Tek contains sev- These data also are consistent with the possibility that eral cell adhesion motifs that are known to play roles in Tek is responsible for directly mediating the endocar- cellular motility, cell recognition, and/or ligand binding dial/myocardial interactions. These interactions could (Bevilacqua 1993). The virtually identical phenotype of

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Dumont et al.

Figure 7. Endothelial cells in the yolk sac of tek asp homozygous embryos express low levels of the tek-lacZ transgene. Thin sec- tions taken from the yolk sacs presented in Fig. 5 illustrate tek promoter-lacZ expres- sion (arrowheads) in the endothelial cells of E8.5 (A,C) and E9.0 (B,D) tek asp homozy- gous (C,D) and wild-type (A,B) embryos. These photomicrographs show both a re- duction in the number of blue-staining en- dothelial cells and a decrease in the levels of [3-galactosidase activity in the mutants. In addition, increased blood cell number can be seen in the blood vessels of tek asp homozygous embryos. Bars, 25 ~m (A,C); 12.5 txm (B,D).

the most extreme Tek-DN mutant and the targeted null stein 1991; Schlessinger and Ullrich 1992). Whether Tek tek asp homozygous embryos demonstrates that expres- and Tie bind similar ligands as heterodimers and/or ho- sion of a catalytically inactive receptor is not sufficient modimers is not known as their ligands have not been for normal Tek function; rather, these data argue that identified. Furthermore, the extracellular domains of the intrinsic tyrosine kinase of the Tek receptor is oblig- these RTKs share a common overall structure; however, atory for development. their primary amino acid sequence homology is low, sug- gesting that Tek and Tie bind distinct ligands. Moreover, Do Tek and Tie heterodimerize? the similar phenotype of dominant-negative and mutant homozygous tek asp embryos suggests that Tek signals as The dimerization and heterodimerization of RTKs upon a homodimer, although the possibility that Tek and Tie ligand binding has been well established (Reith and Bern- signal as obligate heterodimers cannot be excluded.

Table 3. The ratio of lacZ-positive endothelial cells to endoderm cells in the yolk sacs of embryos of F1 intercrosses of tek-lacZ/tek-lacZ;tek~Sv/+ mice

Total no. of Total no. of No. of lacZ + Gestational age tek endoderm cells lacZ+-expressing cells cells per 100 (days) Genotype per blood island a per blood island endoderm cells 8.5 +/- 7.8 _+ 0.9 (24) 4.5 _+ 0.7 54 + 12 8.5 / 6.1 + 1.1 (45) 1.0 _+ 0.8 35---7 9.0 +/- 11.8 -+ 0.3 (27) 4.7 -+ 0.6 39__+6 9.0 / 11.8 _+ 3 (21) 1.1 _+ 0.9 8_+4 aNumbers reflect the mean --- S.D., and the number in brackets represents the number of blood islands counted per section.

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Vascular detects in tek-deficient mice

Are the Tek and Tie signaling pathways redundant? quences is not known. DNAs from these constructs were pre- pared and injected into fertilized oocytes, as described previ- tek asp homozygous embryos express tie at the normal ously (Logan et al. 1993). Embryos were analyzed on days 9.5 time, demonstrating that tie expression is not dependent and 10.5 postinjection and were genotyped by PCR analysis on tek. The finding that tie is expressed in homozygous of yolk sac DNA prepared as described (Frohman et al. 1990), tek asp mutants suggests that the closely related Tie re- using a primer that annealed within the tek 3'-untranslated se- ceptor is unable to compensate for the lack of Tek. How- quence (CCTCACCTGCAGAAGCCAGTTTGT) and primers ever, tie is normally expressed half a day later than tek within either the SV40 (GTGGTTTGTCCAACTGATCAATG) during development (D. Dumont, G.-H. Fong, M. Puri, or polyoma (CTACCATAATCCAGTCTACTGC) poly(A) G. Gradwohl, K. Alitalo, and M. Breitman, in prep.I. sequences. Transgene expression levels could not be ascertained by RNA Thus, if even these two RTKs have completely overlap- in situ analysis using probes directed against the viral poly(A) ping signaling pathways, their different temporal pat- sequences, suggesting that either they were not used and that terns of expression may preclude simple functional re- the tek poly(A) sequences within the tek cDNA were used or dundancy in a dying embryo. Alternatively, Tie and Tek that the levels were too low to be detected. may not bind the same ligand; thus, Tie would not be able to transduce a signal intracellularly in response to tek targeting vector the Tek ligand. It is also possible that Tek and Tie are The tek genomic clone used in these studies was obtained from activated by the same ligand but have distinct down- a 129Sv mouse strain library. The targeting vector consisted of stream signaling pathways as a result of differences in a long arm 7.2-kb Asp 718I-BglII genomic fragment located 5' of their kinase insert and carboxy tail (Dumont et al. 1993). the tek-coding sequences and a short arm of 0.7 kb extending The Tek-DN phenotype is very similar to the homozy- from XbaI to the EcoRI sites immediately 3' of the first exon gous tek asp mutant phenotype, supporting the idea that (Fig. 3). These two fragments were cloned on either side of the the Tek-DN protein is interfering specifically with only phosphoglycerate kinase (PGK)-neo expression cassette of the the Tek signaling pathway. pPNT vector (Tybulewicz et al. 1991) such that the direction of neo transcription was in the same orientation as tek. Upon ho- In summary, we have presented genetic evidence that mologous recombination, this vector will delete -0.7 kb of ge- Tek, one of five known RTKs whose expression is pri- nomic sequences that includes 14 bp of untranslated sequence, marily restricted to endothelial cells, is absolutely re- the first 52 nucleotides of the protein-coding sequence, and quired in the mouse for normal development of the vas- -650 bp of the first intron. culature. Our data argue that Tek function is not re- quired for the initial appearance of early endothelial cells Generation and genotyping of tek asp mice but, rather, for their subsequent survival and/or prolif- R1 (Nagy et al. 1993) ES cells were propagated, electroporated, eration. It will be interesting to determine whether any plated, and selected as described (Joyner et al. 1989). Selection in of the other four RTKs play a critical role in the early gancyclovir resulted in an enrichment of 7- and 32-fold in the developmental decisions to give rise to this important two experiments. Four targeted clones were identified (1 in 232 lineage. and 3 in 55, respectively). Taken together, the frequency of ho- mologous recombination was -1 in 960 G418 R clones. The identification of targeted events was accomplished by Southern Materials and methods blot analysis on ES cell DNA extracted directly in 24-well cul- ture dishes as described (Wurst and Joyner 1993) and digested Generation of the tekAasa dominant-negative transgenes with BglII. A 0.3-kb AccI-BglII genomic DNA fragment located and transgenic embryos immediately 3' to the short arm was used as probe. This probe The codon for lysine-853 was altered by oligonucleotide-di- recognizes a wild-type fragment of 2.5 kb and a targeted frag- rected mutagenesis (Amersham) to the codon encoding an ala- ment of 1.9 kb {Fig. 3B). Confirmation of a correctly targeted nine residue. The entire cDNA fragment used in this mutagen- event was accomplished by Southern analysis of DNA extracted esis was sequenced completely before subcloning back into the from heterozygous mice and digested with multiple enzymes. full-length tek eDNA. The mutated cDNA (tek Ass3) was cloned The probes used were the 3' external and two other internal into the mammalian expression vector pECE (Ellis et al. 1987) probes consisting of the neo-coding sequences and a genomic and transfected into COS cells as described (Dumont et al. DNA fragment of 0.4 kb (SpeI-BglII) found 5' to the protein- 1993). Metabolic labeling and tyrosine kinase assays were done coding sequences (data not shownl. No nonrepetitive probes with an anti-Tek antibody as described (Lhotak and Pawson could be found 5' of the Asp718I site. Injection of ES cells car- 1993). Two of the three transgenes were made by cloning the rying the tek asp mutation into C57BL/6J blastocysts was per- tek Ass3 eDNA upstream of the SV40 polyadenylation [poly(A)] formed as described previously (Joyner et al. 1989). Genotyping sequences (BamHI-XbaI) and then cloning this cassette down- of offspring was carried out on DNA extracted from either tails stream of the large [3-actin promoter (gift of V. Giguiere, Hos- or the dissected heads of embryos. The tek-lacZ transgenic pital for Sick Children, Toronto, Canada) or the 7.2-kb tek pro- mouse line is described {M.C. Puri, D.J. Dumont, and M. Breit- moter (M. Puri, D. Dumont, and M. Breitman, in prep.). The man, in prep). Genotyping of lacZ transgenic animals was de- polyoma promoter-driven transgene was constructed by cloning termined by Southern analysis using lacZ-coding sequences as the tek ASs3 eDNA without the SV40 poly(A) sequences into probe. pdPX13Bla3MTs (Bautch et al. 1987) in which the sequences Mice heterozygous for the tek asp mutation had no apparent coding for polyoma middle T- had been removed by abnormalities and were fertile. Intercrosses of mice derived BstXI digestion. These transgenes all contained 3'-untranslated from both independent ES cell clones were carried out between sequences from the tek cDNA; thus, whether transcription ter- either outbred (129SvJxC57BL/6J) F1 or inbred 129SvJ F 1 mice minated at the tek poly{A) sequences or the viral poly(A) se- to allow analysis on two genetic backgrounds. No differences in

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Dumont et al. phenotype were observed on either of the two genetic back- M.L. Breitman. 1992. tek, a novel tyrosine kinase gene lo- grounds or the two targeted ES cell lines. cated on mouse chromosome 4, is expressed in endothelial cells and their presumptive precursors. Oncogene 7: 1471- 1480. Histology and lacZ staining Dumont, D.J., G.J. Gradwohl, G.-H. Fong, R. Auerbach, and Midday of the vaginal plug was considered as day 0.5 postcoi- M.L. Breitman. 1993. The endothelial-specific receptor tyro- turn in the staging of embryos. To date, all embryos with a sine kinase, tek, is a member of a new subfamily of recep- cobblestone-like appearing yolk sac were homozygous for the tors. Oncogene 8: 1293-1301. tek asp mutation. Therefore, to conserve material, embryos used Eichmann, A., C. Marcelle, C. Br6ant, and N.M. Le Douarin. in the lacZ expression studies were judged to be homozygous for 1993. Two molecules related to the VEGF receptor are ex- the tek Asp mutation based on this criteria. Staining for the pres- pressed in early endothelial cells during avian embryonic ence of ~-gal in whole-mount embryos was performed as de- development. Mech. Dev. 42: 33-48. scribed (Logan et al. 1993). Stained embryos were postfixed in Ellis, L., D.O. Morgna, S.M. Jong, L.H. Wang, R.A. Roth, and formalin at room temperature overnight and processed for wax W.J. Rutter. 1987. Heterologous transmembrane signalling embedding, sectioned at 6 ~m, and counterstained with nu- by a human receptor-v-ros hybrid in Chinese ham- clear-fast red. Quantification of the number of lacZ-expressing ster ovary cells. Proc. Natl. Acad. Sci. 84: 5101-5105. (blue) endothelial cells was accomplished by selecting a single Frohman, M.A., M. Boyle, and G.R. Martin. 1990. Isolation of section of an embryo and counting the number of endoderm and the mouse Hox-2.9 gene; analysis of embryonic expression blue endothelial cells per blood island. Subsequent histological suggests that positional information along the anterior-pos- analysis of these mutants revealed other abnormalities charac- terior axis is specified by mesoderm. Development 110: 589- teristic of homozygous mutants that confirmed the phenotyp- 607. ing. For histological and RNA in situ analysis, the heads of Hunter, T. and R.A. Lindberg. 1994. Receptor protein-tyrosine embryos were removed for DNA extraction and genotyping be- kinases. Frontiers Mol. Biol. (in press). fore fixing the embryos overnight in freshly prepared 4% Iwama, A., I. Hamaguchi, M. Hashiyama, Y. Murayama, K. Ya- paraformaldehyde at 4~ After fixation embryos were pro- sunaga, and T. Suda. 1993. Molecular cloning and character- cessed for wax embedding, sectioned at 4-6 ~m, and either used ization of mouse tie and tek receptor tyrosine kinase genes for RNA in situ analysis or stained with hematoxylin-eosin. and their expression in hematopoietic stem cells. Biochem. Bioph. Res. Comm. 195: 301-309. Joyner, A.L., W.C. Skames, and J. Rossant. 1989. Production of Acknowledgments a mutation in mouse En-2 gene by homologous recombina- We gratefully acknowledge K. Harpal for preparation and sec- tion in embryonic stem cells. Nature 338: 153-156. tioning all embryonic tissues; Dr. W. Wurst for his invaluable Kaipainen, A., J. Korhonen, K. Pajusola, O. Aprelikova, M.G. instruction to D.J.D. on ES cell culture; Dr. K. Alitalo for the tie Perisco, B.L. Torman, and K. Afitalo. 1994. The related FLT4, probe; Dr. J. Hassel for pdPX~gBla3MTs; Drs. A. Bernstein and A. FLT1 and KDR receptor tyrosine kinases show distinct ex- Joyner for critical reading of the manuscript; the gene targeting pression patterns in human fetal endothelial cells. J. Exp. group for helpful discussions; and Z. Krzyzek for excellent sec- Med. 178: 2077-2088. retarial assistance. This work was supported by Bristol Myers-- Kaufman, M.H. and V. Navaratnam. 1981. Early differentiation Squibb and the National Cancer Institute of Canada. M.C.P. is of the heart in mouse embryos. I. Anat. 133: 235-246. an Ontario graduate scholar. The late M.L.B. was an MRC Lhotak, V. and T. Pawson. 1993. Biological and biochemical scientist. activities of a chimeric epidermal growth factor-Elk receptor The publication costs of this article were defrayed in part by tyrosine kinase. Mol. Cell. Biol. 13: 7071-7079. payment of page charges. This article must therefore be hereby Logan, C., W.K. Khoo, D. Cado, and A.L. Joyner. 1993. 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Dominant-negative and targeted null mutations in the endothelial receptor tyrosine kinase, tek, reveal a critical role in vasculogenesis of the embryo.

D J Dumont, G Gradwohl, G H Fong, et al.

Genes Dev. 1994, 8: Access the most recent version at doi:10.1101/gad.8.16.1897

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